RESEARCH ARTICLE
Intragenic Duplication—A Novel Causative Mechanism for SATB2-Associated Syndrome Agne Liede´n,1,2,3* Malin Kvarnung,1,2,3 Daniel Nilssson,1,2,3 Ellika Sahlin,1,2,3 and Elisabeth Syk Lundberg1,2,3 1
Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden
2
Center for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden Department of Clinical Genetics, Karolinska University Hospital, Stockholm, Sweden
3
Manuscript Received: 9 February 2014; Manuscript Accepted: 1 August 2014
Previous studies have shown that genetic aberrations involving the special AT-rich sequence-binding protein 2 (SATB2) gene result in a variable phenotype of syndromic intellectual disability. Although only a small number of patients have been described, there is already considerable variation in regard to the underlying molecular mechanism spanning from structural variation to point mutations. We here describe a male patient with intellectual disability, speech and language impairment, cleft palate, malformed teeth, and oligodontia. Array CGH analysis identified a small intragenic duplication in the SATB2 gene that included three coding exons. The result was confirmed by multiplex ligation-dependent probe amplification and low coverage whole genome mate pair sequencing. WGS breakpoint analysis directly confirmed the duplication as intragenic. This is the first reported patient with an intragenic duplication in SATB2 in combination with a phenotype that is highly similar to previously described patients with small deletions or point mutations of the same gene. Our findings expand the spectra of SATB2 mutations and confirm the presence of a distinct SATB2-phenotype with severe ID and speech impairment, cleft palate and/or high arched palate, and abnormalities of the teeth. For patients that present with this clinical picture, a high-resolution exon targeted array CGH and/or WGS, in addition to sequencing of SATB2, should be considered. Ó 2014 Wiley Periodicals, Inc.
Key words: SATB2; 2q33.1; cleft palate; teeth abnormality; intellectual disability; speech impairment; array comparative genomic hybridization; mate pair sequencing
INTRODUCTION The special AT-rich sequence-binding protein 2 (SATB2) gene (OMIM#608148) encodes a protein involved in chromatin remodeling and transcriptional regulation. [Britanova et al., 2005; Szemes et al., 2006]. The association with human disease has been known for more than a decade, since the identification of de novo balanced translocations with breakpoints intersecting or bordering the gene SATB2 on chromosome 2q33.1 in two patients suffering from mild
Ó 2014 Wiley Periodicals, Inc.
How to Cite this Article: Liede´n A, Kvarnung M, Nilssson D, Sahlin E, Lundberg ES. 2014. Intragenic duplication—A novel causative mechanism for SATB2-associated syndrome. Am J Med Genet Part A 164A:3083–3087.
intellectual disability (ID) and cleft palate [Brewer et al., 1999; FitzPatrick et al., 2003]. Genetic defects affecting SATB2 have succeedingly been reported in a number of patients with somewhat variable phenotypes including ID, seizures, abnormal behavior, cleft palate, micrognathia, tooth abnormalities, slender body habitus, bowing of the tibiae, and osteoporosis [Leoyklang et al., 2007; Rosenfeld et al., 2009; Tegay et al., 2009; Balasubramanian et al., 2011; Rauch et al., 2012; Talkowski et al., 2012; Docker et al., 2013; Rainger et al., 2014]. The reported SATB2 gene defects are diverse despite a limited number of patients. They can be divided into structural defects such as balanced translocations, point mutations, and intragenic deletions. In addition there are patients with deletions or duplications that include SATB2 and also extend further with possible effects on other genes [Van Buggenhout et al., 2005; Urquhart et al., 2009; Usui et al., 2013]. For the first time, we here report on a male patient with an intragenic SATB2 duplication and a phenotype that overlaps previously described patients with deletions or point mutations in SATB2.
Agne Liede´n and Malin Kvarnung contributed equally to this work. Grant sponsor: Swedish Medical Research Council; Grant sponsor: Stockholm County Council. Correspondence to: Agne Liede´n, PhD and Malin Kvarnung MD, Department of Molecular Medicine and Surgery, Karolinska Institutet, 171 76 Stockholm, Sweden E-mail: [email protected] (A.L.); [email protected] (M.K.) Article first published online in Wiley Online Library (wileyonlinelibrary.com): 23 September 2014 DOI 10.1002/ajmg.a.36769
3083
3084
CLINICAL REPORT The proband is a 20-year-old male diagnosed with moderate-severe intellectual disability (ID). He is the only child born to nonconsanguineous healthy parents originating from Sweden and Portugal with no family history of ID. He was born in gestational week 33 after an uneventful pregnancy and with birth parameters within the normal range. Developmental milestones were not met in time; he walked independently at 2 years of age and did not develop any spoken language. A diagnosis of moderate-severe ID with speech and language impairment was later established, and at present age he speaks only a few words and uses sign language for communication. Neurologic examination revealed impairment of fine and gross motor skills, left-sided mild hemiparesis, and spasticity with increased reflexes in the upper and lower extremities bilaterally. MRI of the brain at the age of three years was normal. There is no history of seizures or epilepsy. During childhood and adolescence he has had multiple fractures including clavicle and forearm. Radiological investigation of the spine at the age of 20 years has shown an aberrant pattern consistent with mild-moderate osteoporosis. Furthermore, the patient has abnormalities of the jaw and teeth. Cleft palate was noted early, with surgical correction at the age of 11 months. He has a high arched palate and narrow maxilla with malocclusion. Eruption of the primary as well as of the permanent
AMERICAN JOURNAL OF MEDICAL GENETICS PART A teeth was late and he also has oligodontia. The teeth are morphologically abnormal with conical front teeth, generally large teeth crowns and with extremely short, malformed roots (Fig. 1E). His body habitus is slender with bowing of the tibiae and he exhibits a forward leaning posture with slightly flexed knees and flat feet (Fig. 1C,D). Also noted were broad thumbs and halluces (Fig. 1F,G). Examination for scoliosis was negative. Facial features include a tall forehead, bushy eyebrows and a prominent nose. The facial expression during examination was somewhat striking with peering eyes and a joyful demeanor (Fig. 1A,B). Onset and course of puberty has been within the normal range. His personality, as described by the parents and noted at examination, is seemingly happy and nice with no apparent behavioral problems. Genetic analyses regarding gross chromosome abnormalities, 22q11 deletion, fragile X syndrome and Rubinstein–Taybi had been performed prior to the array CGH analysis with negative results.
MATERIALS AND METHODS Genomic DNA was isolated from whole blood using a standard protocol. Array CGH analysis was performed using the 180K SurePrint G3 Human CGH oligonucleotide microarray (AMAID 022060, Agilent Technologies, Santa Clara, CA) with whole genome coverage and an overall median probe spacing of 13 KB. Labeling of
FIG. 1. Photographs and dental radiography of the patient at the age of 20 years. Facial appearance of the proband showing a tall forehead, a prominent nose, bushy eyebrows, and peering eyes (A and B). Note the slender body habitus, bowing of the tibiae and forward leaning posture with slightly flexed knees and hands (C and D). Panoramic dental radiography showing conical front teeth, generally large teeth crowns with extremely short, malformed roots, oligodontia with agenesis of ten teeth and retention of three molars. One front tooth is replaced with an implant and ceramic crown (E). Photos of the hands and feet exhibiting broad digits and halluces (F and G).
LIEDE´N ET AL. genomic DNA from the patient and reference DNA (Promega Corporation, Madison, WI) and hybridization were performed following the protocols provided by Agilent Technologies. Microarray processing was carried out according to the manufacturer’s recommendations. Scanning of the array slide was done on an Agilent microarray scanner with 3 mm resolution. Data analysis was performed with the Feature Extraction software v10.7.3.1 (Agilent Technologies) and the CytoSure Interpret Software v 3.3.2 (Oxford Gene Technology, Oxfordshire, UK). Array CGH quality control metrics was checked to comply with recommended guidelines (Agilent Technologies). Synthetic oligonucleotides for Multiplex ligation-dependent probe amplification (MLPA) analysis were designed targeting unique sequences within exons three to seven in the SATB2 gene and several control regions according to published recommendations [Stern et al., 2004]. MLPA probes were ordered from MRCHolland (Amsterdam, Noord-Holland, NL) and analysis was carried out according to the manufacturer’s protocol. Pooled genomic DNA (Promega Corporation), were used as control DNA for population based normalization as well as control probe normalization. Amplification products were identified and quantified on an ABI 3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA) and the accompanying software. The tracing data were analyzed in Excel (Microsoft Corporation, Redmond, WA). Massively parallel sequencing was carried out at the Science for Life Laboratory, Stockholm. Briefly, libraries were prepared using Illumina’s Nextera Mate Pair Sample Preparation Kit, according to manufacturer’s instruction, for a gel-free preparation of 2.6 kb effective insert size library (size distribution mode 2.6 kb) and sequenced on an Illumina 2500 sequencer, 2 100 bp. Following Illumina guidelines for Mate Pair post processing (http://res. illumina.com/ documents/products/technotes/technote_nextera_matepair_data_ processing.pdf) adapters sequences were removed using Trimmomatic v0.32 [Bolger et al., 2014]. Remaining pairs have been aligned to
3085 the hg19 human reference genome sequence using bwa 0.7.4-r385 [Li and Durbin, 2009] resulting in a 70 M trimmed uniquely mapping read pairs, with 67.4 M RF read pairs with a mean insert size of 2645 nt and mean mapped coverage of 4.4. Primers were designed for amplification and sequencing of the proximal and distal breakpoints regions and junction analysis indicated by the mate pair sequencing. The primers included: ProxF50 -CAGACAAACTTACAACATAGGC-30 , ProxR 50 -GTACTAAAGTGGTTAAGTGGGT- 30 and DistF 50 -CCAAGTCACCCACACTCAGT- 30 , DistR 50 -GAGATGGGATTTTGTCATGTTGC30 . PCR amplification and Sanger sequencing, performed on an ABI 3730 genetic analyzer (Applied Biosystems) was performed using standard protocols. The study was approved by the Research Ethics Committee at Karolinska Institutet.
RESULTS The array CGH analysis identified a small intragenic duplication in the SATB2 gene. The minimum region contained coding exons but was only supported by three array probes (A_16_P36028520, A_14_P129560, and A_16_P15995191), and the finding was therefore further investigated by MLPA analysis. Three MLPA probes in the region confirmed the presence of a duplication in two independent samples from the patient. The combined results of the array CGH and MLPA analysis show that exon 5, 6, and 7 are included in the duplication region, minimum region chr2:200, 233, 354–200, 255, 458 (GRCh37/hg19) (Fig. 2). Analysis of DNA from parental blood samples showed a normal copy number for the region, suggesting that the duplication has occurred de novo. Several other copy number variations were detected in the array CGH analysis but all of them were in regions of known normal variation.
FIG. 2. Intragenic SATB2 duplication. Combined results of array CGH, MLPA probes and sequencing in the duplication region. Filled black circles represents array CGH probes and empty white circles are MLPA probes. The results of the analyzed probes are shown as normalized log2 ratio values on the y-axis. Grey block arrows corresponds to the flanking breakpoint regions and connecting lines orientation as indicated by WGS analysis. Positions are shown in relation to the genomic position on chromosome 2 (on top) in GRCh37/hg19 and also in relation to exons and introns (drawn to real relative size) in transcript NM_015265. Breakpoint sequence analysis of the duplication is shown at the bottom. Proximal reference sequence and patient breakpoint sequences that match are shown with grey background and distal reference sequence and patient breakpoint sequences that match are shown with white letters on black background. Boxed sequence corresponds to microhomology between the distal and proximal sequences.
3086 Low coverage WGS using mate pairs confirmed a heterozygous duplication in tandem, encompassing the region chr2:200, 228, 800–200, 263, 582. This directly shows that the duplication is intragenic, with intronic breakpoints. Breakpoint junction analysis show that three base pairs (CAC) of microhomology are shared between the proximal and distal reference sequences and that the distal breakpoint maps within a short local repeat. The final result from the fine mapping show that the duplicated segment corresponds to the region chr2:200, 228, 772–200, 263, 785 (35 kb). As expected, using the distal forward primer and the proximal reverse primer produced a 700 bp amplification product for the patient that was not detected when analyzing the parental samples (gel picture not shown).
DISCUSSION We here report on a patient with an intragenic duplication in SATB2 and a phenotype overlapping previously described patients with isolated SATB2 aberrations. To the best of our knowledge, no other similar patients have been published in the literature, although two additional patients with intragenic duplications can be found in the ISCA database (www.iscaconsortium.org.) and a third has been described in a conference abstract [Kaiser et al., 2013]. Breakpoint junction analysis show microhomology at the breakpoint and may therefore support a replicative mechanism such as fork stalling and template switching/microhomology-mediated breakage-induced replication as the underlying mechanism for the generation of the duplication [Zhang et al., 2009]. Genetic aberrations involving SATB2 is a known cause of syndromatic ID with additional symptoms such as cleft palate. Different types of genetic defects have been reported and can be divided into isolated SATB2 defects and aberrations that may affect other genes in addition to SATB2. The latter group includes deletions that encompass several genes as well as balanced translocations and the phenotype of these patients is somewhat diverse, possibly due to the involvement of different genes in addition to SATB2. Isolated defects restricted to SATB2 have been described in a few patients and include aberrations such as point mutations or small deletions. To date, six detailed clinical descriptions of such patients have been reported (two with point mutations and four with deletions), all with a phenotype very similar to the one of our patient [Leoyklang et al., 2007; Rosenfeld et al., 2009; Balasubramanian et al., 2011; Docker et al., 2013]. All patients, including the present, exhibit severe ID with absent or nearly absent speech, normal MRI of the brain, similar facial features and abnormalities of the teeth. Common facial features include micrognathia, a long face, a broad or tall forehead and a prominent nose or nasal bridge. Cleft palate was present in four out of six previous patients as well as in our patient. Our findings of tibial bowing and osteoporosis were reported previously by Leoyklang et al. in a patient with a point mutation in SATB2. Findings of spasticity and hemiparesis, as seen in our patient, has not been reported in previous cases, even though one of the patients reported by Rosenfeld et al. exhibited muscular hypertonia and increased deep tendon reflexes. We can not conclude whether the finding of spastic hemiparesis in our patient is part of the syndromic phenotype or secondary to other factors such as prematurity. In summary, the phenotype of our patient is very
AMERICAN JOURNAL OF MEDICAL GENETICS PART A similar to the phenotype of previously described patients with small deletions or point mutations in SATB2. So far, no features have been delineated that may be used to clearly distinguish patients with point mutations, deletions or duplications in SATB2. This observation suggests that the phenotype of these patients may be caused by a common deficiency of functional SATB2 protein, even though the underlying molecular mechanisms may differ depending on the type of SATB2 aberration. Leoyklang et al. [2013] recently suggested that mutations in SATB2, which preserve the dimerization domain of the protein, cause disease due to a dominant negative effect as opposed to deletions, which lead to symptoms via haploinsufficiency. One may expect a more severe phenotype in patients with genetic defects that have a dominant negative effect as compared to patients with haploinsufficiency due to deletions. As noted earlier, no differentiating phenotypic features were seen in our patient when compared to the six previously described patients, but the number of reported patients is still too small to reliably establish or exclude the presence of a genotype-phenotype correlation within the group of isolated SATB2 defects. In conclusion, our results demonstrate the first patient with an intragenic duplication in SATB2 causing a phenotype that is highly similar to previously described patients with small deletions or point mutations of the same gene. Our findings expand the spectrum of SATB2 mutations and confirm the presence of a distinct SATB2-phenotype with severe ID and speech impairment, cleft palate and/or high arched palate, normal brain MRI, abnormalities of the teeth, and common facial features. For patients that present with this clinical picture, a high-resolution exon targeted array CGH and/or WGS, in addition to sequencing of SATB2, should be considered.
ACKNOWLEDGMENTS We thank the patient and his parents for participating in the study. We acknowledge support from Science for Life Laboratory, the national infrastructure SNISS, and Uppmax for providing assistance in massive parallel sequencing and computational infrastructure. More specifically, we acknowledge Francesco Vezzi och Birgitta Lo¨tstedt for excellent technical assistance. We are also grateful to Dr John-Erik Nyman, licensed dental surgeon, pedodontist and former senior consultant in pedodontics for describing the patient’s dental phenotype. The study was funded by grants from the Swedish Medical Research Council and the Stockholm County Council.
REFERENCES Balasubramanian M, Smith K, Basel-Vanagaite L, Feingold MF, Brock P, Gowans GC, Vasudevan PC, Cresswell L, Taylor EJ, Harris CJ, Friedman N, Moran R, Feret H, Zackai EH, Theisen A, Rosenfeld JA, Parker MJ. 2011. Case series: 2q33.1 microdeletion syndrome–further delineation of the phenotype. J Med Genet 48:290–298. Bolger AM, Lohse M, Usadel B. 2014. Trimmomatic: A flexible trimmer for Illumina sequence data. Bioinformatics 30:2114–2120. Brewer CM, Leek JP, Green AJ, Holloway S, Bonthron DT, Markham AF, FitzPatrick DR. 1999. A locus for isolated cleft palate, located on human chromosome 2q32. Am J Hum Genet 65:387–396.
LIEDE´N ET AL. Britanova O, Akopov S, Lukyanov S, Gruss P, Tarabykin V. 2005. Novel transcription factor Satb2 interacts with matrix attachment region DNA elements in a tissue-specific manner and demonstrates cell-typedependent expression in the developing mouse CNS. Eur J Neurosci 21:658–668. Docker D, Schubach M, Menzel M, Munz M, Spaich C, Biskup S, Bartholdi D. 2013. Further delineation of the SATB2 phenotype. Eur J Hum Genet 22:1034–1039. FitzPatrick DR, Carr IM, McLaren L, Leek JP, Wightman P, Williamson K, Gautier P, McGill N, Hayward C, Firth H, Markham AF, Fantes JA, Bonthron DT. 2003. Identification of SATB2 as the cleft palate gene on 2q32-q33. Hum Mol Genet 12:2491–2501. Kaiser AS, Maas B, Wolff A, Hinderhofer K, Sutter C, Janssen JWG, Moog U. 2013. Girl with developmental delay, nearly absent speech and oligodontia: First case of an intragenic SATB2 gene duplication. Medizinische Genetik 25:125. Leoyklang P, Suphapeetiporn K, Siriwan P, Desudchit T, Chaowanapanja P, Gahl WA, Shotelersuk V. 2007. Heterozygous nonsense mutation SATB2 associated with cleft palate, osteoporosis, and cognitive defects. Hum Mutat 28:732–738. Leoyklang P, Suphapeetiporn K, Srichomthong C, Tongkobpetch S, Fietze S, Dorward H, Cullinane AR, Gahl WA, Huizing M, Shotelersuk V. 2013. Disorders with similar clinical phenotypes reveal underlying genetic interaction: SATB2 acts as an activator of the UPF3B gene. Hum Genet 132:1383–1393. Li H, Durbin R. 2009. Fast and accurate short read alignment with BurrowsWheeler transform. Bioinformatics 25:1754–1760. Rainger JK, Bhatia S, Bengani H, Gautier P, Rainger J, Pearson M, Ansari M, Crow J, Mehendale F, Palinkasova B, Dixon MJ, Thompson PJ, Matarin M, Sisodiya SM, Kleinjan DA, Fitzpatrick DR. 2014. Disruption of SATB2 or its long-range cis-regulation by SOX9 causes a syndromic form of Pierre Robin sequence. Hum Mol Genet 23:2569–2579. Rauch A, Wieczorek D, Graf E, Wieland T, Endele S, Schwarzmayr T, Albrecht B, Bartholdi D, Beygo J, Di Donato N, Dufke A, Cremer K, Hempel M, Horn D, Hoyer J, Joset P, Ropke A, Moog U, Riess A, Thiel CT, Tzschach A, Wiesener A, Wohlleber E, Zweier C, Ekici AB, Zink AM, Rump A, Meisinger C, Grallert H, Sticht H, Schenck A, Engels H, Rappold G, Schrock E, Wieacker P, Riess O, Meitinger T, Reis A, Strom TM. 2012. Range of genetic mutations associated with severe non-syndromic
3087 sporadic intellectual disability: An exome sequencing study. Lancet 380:1674–1682. Rosenfeld JA, Ballif BC, Lucas A, Spence EJ, Powell C, Aylsworth AS, Torchia BA, Shaffer LG. 2009. Small deletions of SATB2 cause some of the clinical features of the 2q33.1 microdeletion syndrome. PLoS One 4:e6568. Stern RF, Roberts RG, Mann K, Yau SC, Berg J, Ogilvie CM. 2004. Multiplex ligation-dependent probe amplification using a completely synthetic probe set. Biotechniques 37:399–405. Szemes M, Gyorgy A, Paweletz C, Dobi A, Agoston DV. 2006. Isolation and characterization of SATB2, a novel AT-rich DNA binding protein expressed in development- and cell-specific manner in rat brain. Neurochem Res 31:237–246. Talkowski ME, Rosenfeld JA, Blumenthal I, Pillalamarri V, Chiang C, Heilbut A, Ernst C, Hanscom C, Rossin E, Lindgren AM, Pereira S, Ruderfer D, Kirby A, Ripke S, Harris DJ, Lee JH, Ha K, Kim HG, Solomon BD, Gropman AL, Lucente D, Sims K, Ohsumi TK, Borowsky ML, Loranger S, Quade B, Lage K, Miles J, Wu BL, Shen Y, Neale B, Shaffer LG, Daly MJ, Morton CC, Gusella JF. 2012. Sequencing chromosomal abnormalities reveals neurodevelopmental loci that confer risk across diagnostic boundaries. Cell 149:525–537. Tegay DH, Chan KK, Leung L, Wang C, Burkett S, Stone G, Stanyon R, Toriello HV, Hatchwell E. 2009. Toriello-Carey syndrome in a patient with a de novo balanced translocation [46,XY,t(2;14)(q33;q22)] interrupting SATB2. Clin Genet 75:259–264. Urquhart J, Black GC, Clayton-Smith J. 2009. 4.5 Mb microdeletion in chromosome band 2q33.1 associated with learning disability and cleft palate. Eur J Med Genet 52:454–457. Usui D, Shimada S, Shimojima K, Sugawara M, Kawasaki H, Shigematu H, Takahashi Y, Inoue Y, Imai K, Yamamoto T. 2013. Interstitial duplication of 2q32.1-q33.3 in a patient with epilepsy, developmental delay, and autistic behavior. Am J Med Genet A 161A:1078–1084. Van Buggenhout G, Van Ravenswaaij-Arts C, Mc Maas N, Thoelen R, Vogels A, Smeets D, Salden I, Matthijs G, Fryns JP, Vermeesch JR. 2005. The del(2)(q32.2q33) deletion syndrome defined by clinical and molecular characterization of four patients. Eur J Med Genet 48:276–289. Zhang F, Khajavi M, Connolly AM, Towne CF, Batish SD, Lupski JR. 2009. The DNA replication FoSTeS/MMBIR mechanism can generate genomic, genic and exonic complex rearrangements in humans. Nat Genet 41:849–853.
Intragenic Duplication—A Novel Causative Mechanism for SATB2-Associated Syndrome Agne Liede´n,1,2,3* Malin Kvarnung,1,2,3 Daniel Nilssson,1,2,3 Ellika Sahlin,1,2,3 and Elisabeth Syk Lundberg1,2,3 1
Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden
2
Center for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden Department of Clinical Genetics, Karolinska University Hospital, Stockholm, Sweden
3
Manuscript Received: 9 February 2014; Manuscript Accepted: 1 August 2014
Previous studies have shown that genetic aberrations involving the special AT-rich sequence-binding protein 2 (SATB2) gene result in a variable phenotype of syndromic intellectual disability. Although only a small number of patients have been described, there is already considerable variation in regard to the underlying molecular mechanism spanning from structural variation to point mutations. We here describe a male patient with intellectual disability, speech and language impairment, cleft palate, malformed teeth, and oligodontia. Array CGH analysis identified a small intragenic duplication in the SATB2 gene that included three coding exons. The result was confirmed by multiplex ligation-dependent probe amplification and low coverage whole genome mate pair sequencing. WGS breakpoint analysis directly confirmed the duplication as intragenic. This is the first reported patient with an intragenic duplication in SATB2 in combination with a phenotype that is highly similar to previously described patients with small deletions or point mutations of the same gene. Our findings expand the spectra of SATB2 mutations and confirm the presence of a distinct SATB2-phenotype with severe ID and speech impairment, cleft palate and/or high arched palate, and abnormalities of the teeth. For patients that present with this clinical picture, a high-resolution exon targeted array CGH and/or WGS, in addition to sequencing of SATB2, should be considered. Ó 2014 Wiley Periodicals, Inc.
Key words: SATB2; 2q33.1; cleft palate; teeth abnormality; intellectual disability; speech impairment; array comparative genomic hybridization; mate pair sequencing
INTRODUCTION The special AT-rich sequence-binding protein 2 (SATB2) gene (OMIM#608148) encodes a protein involved in chromatin remodeling and transcriptional regulation. [Britanova et al., 2005; Szemes et al., 2006]. The association with human disease has been known for more than a decade, since the identification of de novo balanced translocations with breakpoints intersecting or bordering the gene SATB2 on chromosome 2q33.1 in two patients suffering from mild
Ó 2014 Wiley Periodicals, Inc.
How to Cite this Article: Liede´n A, Kvarnung M, Nilssson D, Sahlin E, Lundberg ES. 2014. Intragenic duplication—A novel causative mechanism for SATB2-associated syndrome. Am J Med Genet Part A 164A:3083–3087.
intellectual disability (ID) and cleft palate [Brewer et al., 1999; FitzPatrick et al., 2003]. Genetic defects affecting SATB2 have succeedingly been reported in a number of patients with somewhat variable phenotypes including ID, seizures, abnormal behavior, cleft palate, micrognathia, tooth abnormalities, slender body habitus, bowing of the tibiae, and osteoporosis [Leoyklang et al., 2007; Rosenfeld et al., 2009; Tegay et al., 2009; Balasubramanian et al., 2011; Rauch et al., 2012; Talkowski et al., 2012; Docker et al., 2013; Rainger et al., 2014]. The reported SATB2 gene defects are diverse despite a limited number of patients. They can be divided into structural defects such as balanced translocations, point mutations, and intragenic deletions. In addition there are patients with deletions or duplications that include SATB2 and also extend further with possible effects on other genes [Van Buggenhout et al., 2005; Urquhart et al., 2009; Usui et al., 2013]. For the first time, we here report on a male patient with an intragenic SATB2 duplication and a phenotype that overlaps previously described patients with deletions or point mutations in SATB2.
Agne Liede´n and Malin Kvarnung contributed equally to this work. Grant sponsor: Swedish Medical Research Council; Grant sponsor: Stockholm County Council. Correspondence to: Agne Liede´n, PhD and Malin Kvarnung MD, Department of Molecular Medicine and Surgery, Karolinska Institutet, 171 76 Stockholm, Sweden E-mail: [email protected] (A.L.); [email protected] (M.K.) Article first published online in Wiley Online Library (wileyonlinelibrary.com): 23 September 2014 DOI 10.1002/ajmg.a.36769
3083
3084
CLINICAL REPORT The proband is a 20-year-old male diagnosed with moderate-severe intellectual disability (ID). He is the only child born to nonconsanguineous healthy parents originating from Sweden and Portugal with no family history of ID. He was born in gestational week 33 after an uneventful pregnancy and with birth parameters within the normal range. Developmental milestones were not met in time; he walked independently at 2 years of age and did not develop any spoken language. A diagnosis of moderate-severe ID with speech and language impairment was later established, and at present age he speaks only a few words and uses sign language for communication. Neurologic examination revealed impairment of fine and gross motor skills, left-sided mild hemiparesis, and spasticity with increased reflexes in the upper and lower extremities bilaterally. MRI of the brain at the age of three years was normal. There is no history of seizures or epilepsy. During childhood and adolescence he has had multiple fractures including clavicle and forearm. Radiological investigation of the spine at the age of 20 years has shown an aberrant pattern consistent with mild-moderate osteoporosis. Furthermore, the patient has abnormalities of the jaw and teeth. Cleft palate was noted early, with surgical correction at the age of 11 months. He has a high arched palate and narrow maxilla with malocclusion. Eruption of the primary as well as of the permanent
AMERICAN JOURNAL OF MEDICAL GENETICS PART A teeth was late and he also has oligodontia. The teeth are morphologically abnormal with conical front teeth, generally large teeth crowns and with extremely short, malformed roots (Fig. 1E). His body habitus is slender with bowing of the tibiae and he exhibits a forward leaning posture with slightly flexed knees and flat feet (Fig. 1C,D). Also noted were broad thumbs and halluces (Fig. 1F,G). Examination for scoliosis was negative. Facial features include a tall forehead, bushy eyebrows and a prominent nose. The facial expression during examination was somewhat striking with peering eyes and a joyful demeanor (Fig. 1A,B). Onset and course of puberty has been within the normal range. His personality, as described by the parents and noted at examination, is seemingly happy and nice with no apparent behavioral problems. Genetic analyses regarding gross chromosome abnormalities, 22q11 deletion, fragile X syndrome and Rubinstein–Taybi had been performed prior to the array CGH analysis with negative results.
MATERIALS AND METHODS Genomic DNA was isolated from whole blood using a standard protocol. Array CGH analysis was performed using the 180K SurePrint G3 Human CGH oligonucleotide microarray (AMAID 022060, Agilent Technologies, Santa Clara, CA) with whole genome coverage and an overall median probe spacing of 13 KB. Labeling of
FIG. 1. Photographs and dental radiography of the patient at the age of 20 years. Facial appearance of the proband showing a tall forehead, a prominent nose, bushy eyebrows, and peering eyes (A and B). Note the slender body habitus, bowing of the tibiae and forward leaning posture with slightly flexed knees and hands (C and D). Panoramic dental radiography showing conical front teeth, generally large teeth crowns with extremely short, malformed roots, oligodontia with agenesis of ten teeth and retention of three molars. One front tooth is replaced with an implant and ceramic crown (E). Photos of the hands and feet exhibiting broad digits and halluces (F and G).
LIEDE´N ET AL. genomic DNA from the patient and reference DNA (Promega Corporation, Madison, WI) and hybridization were performed following the protocols provided by Agilent Technologies. Microarray processing was carried out according to the manufacturer’s recommendations. Scanning of the array slide was done on an Agilent microarray scanner with 3 mm resolution. Data analysis was performed with the Feature Extraction software v10.7.3.1 (Agilent Technologies) and the CytoSure Interpret Software v 3.3.2 (Oxford Gene Technology, Oxfordshire, UK). Array CGH quality control metrics was checked to comply with recommended guidelines (Agilent Technologies). Synthetic oligonucleotides for Multiplex ligation-dependent probe amplification (MLPA) analysis were designed targeting unique sequences within exons three to seven in the SATB2 gene and several control regions according to published recommendations [Stern et al., 2004]. MLPA probes were ordered from MRCHolland (Amsterdam, Noord-Holland, NL) and analysis was carried out according to the manufacturer’s protocol. Pooled genomic DNA (Promega Corporation), were used as control DNA for population based normalization as well as control probe normalization. Amplification products were identified and quantified on an ABI 3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA) and the accompanying software. The tracing data were analyzed in Excel (Microsoft Corporation, Redmond, WA). Massively parallel sequencing was carried out at the Science for Life Laboratory, Stockholm. Briefly, libraries were prepared using Illumina’s Nextera Mate Pair Sample Preparation Kit, according to manufacturer’s instruction, for a gel-free preparation of 2.6 kb effective insert size library (size distribution mode 2.6 kb) and sequenced on an Illumina 2500 sequencer, 2 100 bp. Following Illumina guidelines for Mate Pair post processing (http://res. illumina.com/ documents/products/technotes/technote_nextera_matepair_data_ processing.pdf) adapters sequences were removed using Trimmomatic v0.32 [Bolger et al., 2014]. Remaining pairs have been aligned to
3085 the hg19 human reference genome sequence using bwa 0.7.4-r385 [Li and Durbin, 2009] resulting in a 70 M trimmed uniquely mapping read pairs, with 67.4 M RF read pairs with a mean insert size of 2645 nt and mean mapped coverage of 4.4. Primers were designed for amplification and sequencing of the proximal and distal breakpoints regions and junction analysis indicated by the mate pair sequencing. The primers included: ProxF50 -CAGACAAACTTACAACATAGGC-30 , ProxR 50 -GTACTAAAGTGGTTAAGTGGGT- 30 and DistF 50 -CCAAGTCACCCACACTCAGT- 30 , DistR 50 -GAGATGGGATTTTGTCATGTTGC30 . PCR amplification and Sanger sequencing, performed on an ABI 3730 genetic analyzer (Applied Biosystems) was performed using standard protocols. The study was approved by the Research Ethics Committee at Karolinska Institutet.
RESULTS The array CGH analysis identified a small intragenic duplication in the SATB2 gene. The minimum region contained coding exons but was only supported by three array probes (A_16_P36028520, A_14_P129560, and A_16_P15995191), and the finding was therefore further investigated by MLPA analysis. Three MLPA probes in the region confirmed the presence of a duplication in two independent samples from the patient. The combined results of the array CGH and MLPA analysis show that exon 5, 6, and 7 are included in the duplication region, minimum region chr2:200, 233, 354–200, 255, 458 (GRCh37/hg19) (Fig. 2). Analysis of DNA from parental blood samples showed a normal copy number for the region, suggesting that the duplication has occurred de novo. Several other copy number variations were detected in the array CGH analysis but all of them were in regions of known normal variation.
FIG. 2. Intragenic SATB2 duplication. Combined results of array CGH, MLPA probes and sequencing in the duplication region. Filled black circles represents array CGH probes and empty white circles are MLPA probes. The results of the analyzed probes are shown as normalized log2 ratio values on the y-axis. Grey block arrows corresponds to the flanking breakpoint regions and connecting lines orientation as indicated by WGS analysis. Positions are shown in relation to the genomic position on chromosome 2 (on top) in GRCh37/hg19 and also in relation to exons and introns (drawn to real relative size) in transcript NM_015265. Breakpoint sequence analysis of the duplication is shown at the bottom. Proximal reference sequence and patient breakpoint sequences that match are shown with grey background and distal reference sequence and patient breakpoint sequences that match are shown with white letters on black background. Boxed sequence corresponds to microhomology between the distal and proximal sequences.
3086 Low coverage WGS using mate pairs confirmed a heterozygous duplication in tandem, encompassing the region chr2:200, 228, 800–200, 263, 582. This directly shows that the duplication is intragenic, with intronic breakpoints. Breakpoint junction analysis show that three base pairs (CAC) of microhomology are shared between the proximal and distal reference sequences and that the distal breakpoint maps within a short local repeat. The final result from the fine mapping show that the duplicated segment corresponds to the region chr2:200, 228, 772–200, 263, 785 (35 kb). As expected, using the distal forward primer and the proximal reverse primer produced a 700 bp amplification product for the patient that was not detected when analyzing the parental samples (gel picture not shown).
DISCUSSION We here report on a patient with an intragenic duplication in SATB2 and a phenotype overlapping previously described patients with isolated SATB2 aberrations. To the best of our knowledge, no other similar patients have been published in the literature, although two additional patients with intragenic duplications can be found in the ISCA database (www.iscaconsortium.org.) and a third has been described in a conference abstract [Kaiser et al., 2013]. Breakpoint junction analysis show microhomology at the breakpoint and may therefore support a replicative mechanism such as fork stalling and template switching/microhomology-mediated breakage-induced replication as the underlying mechanism for the generation of the duplication [Zhang et al., 2009]. Genetic aberrations involving SATB2 is a known cause of syndromatic ID with additional symptoms such as cleft palate. Different types of genetic defects have been reported and can be divided into isolated SATB2 defects and aberrations that may affect other genes in addition to SATB2. The latter group includes deletions that encompass several genes as well as balanced translocations and the phenotype of these patients is somewhat diverse, possibly due to the involvement of different genes in addition to SATB2. Isolated defects restricted to SATB2 have been described in a few patients and include aberrations such as point mutations or small deletions. To date, six detailed clinical descriptions of such patients have been reported (two with point mutations and four with deletions), all with a phenotype very similar to the one of our patient [Leoyklang et al., 2007; Rosenfeld et al., 2009; Balasubramanian et al., 2011; Docker et al., 2013]. All patients, including the present, exhibit severe ID with absent or nearly absent speech, normal MRI of the brain, similar facial features and abnormalities of the teeth. Common facial features include micrognathia, a long face, a broad or tall forehead and a prominent nose or nasal bridge. Cleft palate was present in four out of six previous patients as well as in our patient. Our findings of tibial bowing and osteoporosis were reported previously by Leoyklang et al. in a patient with a point mutation in SATB2. Findings of spasticity and hemiparesis, as seen in our patient, has not been reported in previous cases, even though one of the patients reported by Rosenfeld et al. exhibited muscular hypertonia and increased deep tendon reflexes. We can not conclude whether the finding of spastic hemiparesis in our patient is part of the syndromic phenotype or secondary to other factors such as prematurity. In summary, the phenotype of our patient is very
AMERICAN JOURNAL OF MEDICAL GENETICS PART A similar to the phenotype of previously described patients with small deletions or point mutations in SATB2. So far, no features have been delineated that may be used to clearly distinguish patients with point mutations, deletions or duplications in SATB2. This observation suggests that the phenotype of these patients may be caused by a common deficiency of functional SATB2 protein, even though the underlying molecular mechanisms may differ depending on the type of SATB2 aberration. Leoyklang et al. [2013] recently suggested that mutations in SATB2, which preserve the dimerization domain of the protein, cause disease due to a dominant negative effect as opposed to deletions, which lead to symptoms via haploinsufficiency. One may expect a more severe phenotype in patients with genetic defects that have a dominant negative effect as compared to patients with haploinsufficiency due to deletions. As noted earlier, no differentiating phenotypic features were seen in our patient when compared to the six previously described patients, but the number of reported patients is still too small to reliably establish or exclude the presence of a genotype-phenotype correlation within the group of isolated SATB2 defects. In conclusion, our results demonstrate the first patient with an intragenic duplication in SATB2 causing a phenotype that is highly similar to previously described patients with small deletions or point mutations of the same gene. Our findings expand the spectrum of SATB2 mutations and confirm the presence of a distinct SATB2-phenotype with severe ID and speech impairment, cleft palate and/or high arched palate, normal brain MRI, abnormalities of the teeth, and common facial features. For patients that present with this clinical picture, a high-resolution exon targeted array CGH and/or WGS, in addition to sequencing of SATB2, should be considered.
ACKNOWLEDGMENTS We thank the patient and his parents for participating in the study. We acknowledge support from Science for Life Laboratory, the national infrastructure SNISS, and Uppmax for providing assistance in massive parallel sequencing and computational infrastructure. More specifically, we acknowledge Francesco Vezzi och Birgitta Lo¨tstedt for excellent technical assistance. We are also grateful to Dr John-Erik Nyman, licensed dental surgeon, pedodontist and former senior consultant in pedodontics for describing the patient’s dental phenotype. The study was funded by grants from the Swedish Medical Research Council and the Stockholm County Council.
REFERENCES Balasubramanian M, Smith K, Basel-Vanagaite L, Feingold MF, Brock P, Gowans GC, Vasudevan PC, Cresswell L, Taylor EJ, Harris CJ, Friedman N, Moran R, Feret H, Zackai EH, Theisen A, Rosenfeld JA, Parker MJ. 2011. Case series: 2q33.1 microdeletion syndrome–further delineation of the phenotype. J Med Genet 48:290–298. Bolger AM, Lohse M, Usadel B. 2014. Trimmomatic: A flexible trimmer for Illumina sequence data. Bioinformatics 30:2114–2120. Brewer CM, Leek JP, Green AJ, Holloway S, Bonthron DT, Markham AF, FitzPatrick DR. 1999. A locus for isolated cleft palate, located on human chromosome 2q32. Am J Hum Genet 65:387–396.
LIEDE´N ET AL. Britanova O, Akopov S, Lukyanov S, Gruss P, Tarabykin V. 2005. Novel transcription factor Satb2 interacts with matrix attachment region DNA elements in a tissue-specific manner and demonstrates cell-typedependent expression in the developing mouse CNS. Eur J Neurosci 21:658–668. Docker D, Schubach M, Menzel M, Munz M, Spaich C, Biskup S, Bartholdi D. 2013. Further delineation of the SATB2 phenotype. Eur J Hum Genet 22:1034–1039. FitzPatrick DR, Carr IM, McLaren L, Leek JP, Wightman P, Williamson K, Gautier P, McGill N, Hayward C, Firth H, Markham AF, Fantes JA, Bonthron DT. 2003. Identification of SATB2 as the cleft palate gene on 2q32-q33. Hum Mol Genet 12:2491–2501. Kaiser AS, Maas B, Wolff A, Hinderhofer K, Sutter C, Janssen JWG, Moog U. 2013. Girl with developmental delay, nearly absent speech and oligodontia: First case of an intragenic SATB2 gene duplication. Medizinische Genetik 25:125. Leoyklang P, Suphapeetiporn K, Siriwan P, Desudchit T, Chaowanapanja P, Gahl WA, Shotelersuk V. 2007. Heterozygous nonsense mutation SATB2 associated with cleft palate, osteoporosis, and cognitive defects. Hum Mutat 28:732–738. Leoyklang P, Suphapeetiporn K, Srichomthong C, Tongkobpetch S, Fietze S, Dorward H, Cullinane AR, Gahl WA, Huizing M, Shotelersuk V. 2013. Disorders with similar clinical phenotypes reveal underlying genetic interaction: SATB2 acts as an activator of the UPF3B gene. Hum Genet 132:1383–1393. Li H, Durbin R. 2009. Fast and accurate short read alignment with BurrowsWheeler transform. Bioinformatics 25:1754–1760. Rainger JK, Bhatia S, Bengani H, Gautier P, Rainger J, Pearson M, Ansari M, Crow J, Mehendale F, Palinkasova B, Dixon MJ, Thompson PJ, Matarin M, Sisodiya SM, Kleinjan DA, Fitzpatrick DR. 2014. Disruption of SATB2 or its long-range cis-regulation by SOX9 causes a syndromic form of Pierre Robin sequence. Hum Mol Genet 23:2569–2579. Rauch A, Wieczorek D, Graf E, Wieland T, Endele S, Schwarzmayr T, Albrecht B, Bartholdi D, Beygo J, Di Donato N, Dufke A, Cremer K, Hempel M, Horn D, Hoyer J, Joset P, Ropke A, Moog U, Riess A, Thiel CT, Tzschach A, Wiesener A, Wohlleber E, Zweier C, Ekici AB, Zink AM, Rump A, Meisinger C, Grallert H, Sticht H, Schenck A, Engels H, Rappold G, Schrock E, Wieacker P, Riess O, Meitinger T, Reis A, Strom TM. 2012. Range of genetic mutations associated with severe non-syndromic
3087 sporadic intellectual disability: An exome sequencing study. Lancet 380:1674–1682. Rosenfeld JA, Ballif BC, Lucas A, Spence EJ, Powell C, Aylsworth AS, Torchia BA, Shaffer LG. 2009. Small deletions of SATB2 cause some of the clinical features of the 2q33.1 microdeletion syndrome. PLoS One 4:e6568. Stern RF, Roberts RG, Mann K, Yau SC, Berg J, Ogilvie CM. 2004. Multiplex ligation-dependent probe amplification using a completely synthetic probe set. Biotechniques 37:399–405. Szemes M, Gyorgy A, Paweletz C, Dobi A, Agoston DV. 2006. Isolation and characterization of SATB2, a novel AT-rich DNA binding protein expressed in development- and cell-specific manner in rat brain. Neurochem Res 31:237–246. Talkowski ME, Rosenfeld JA, Blumenthal I, Pillalamarri V, Chiang C, Heilbut A, Ernst C, Hanscom C, Rossin E, Lindgren AM, Pereira S, Ruderfer D, Kirby A, Ripke S, Harris DJ, Lee JH, Ha K, Kim HG, Solomon BD, Gropman AL, Lucente D, Sims K, Ohsumi TK, Borowsky ML, Loranger S, Quade B, Lage K, Miles J, Wu BL, Shen Y, Neale B, Shaffer LG, Daly MJ, Morton CC, Gusella JF. 2012. Sequencing chromosomal abnormalities reveals neurodevelopmental loci that confer risk across diagnostic boundaries. Cell 149:525–537. Tegay DH, Chan KK, Leung L, Wang C, Burkett S, Stone G, Stanyon R, Toriello HV, Hatchwell E. 2009. Toriello-Carey syndrome in a patient with a de novo balanced translocation [46,XY,t(2;14)(q33;q22)] interrupting SATB2. Clin Genet 75:259–264. Urquhart J, Black GC, Clayton-Smith J. 2009. 4.5 Mb microdeletion in chromosome band 2q33.1 associated with learning disability and cleft palate. Eur J Med Genet 52:454–457. Usui D, Shimada S, Shimojima K, Sugawara M, Kawasaki H, Shigematu H, Takahashi Y, Inoue Y, Imai K, Yamamoto T. 2013. Interstitial duplication of 2q32.1-q33.3 in a patient with epilepsy, developmental delay, and autistic behavior. Am J Med Genet A 161A:1078–1084. Van Buggenhout G, Van Ravenswaaij-Arts C, Mc Maas N, Thoelen R, Vogels A, Smeets D, Salden I, Matthijs G, Fryns JP, Vermeesch JR. 2005. The del(2)(q32.2q33) deletion syndrome defined by clinical and molecular characterization of four patients. Eur J Med Genet 48:276–289. Zhang F, Khajavi M, Connolly AM, Towne CF, Batish SD, Lupski JR. 2009. The DNA replication FoSTeS/MMBIR mechanism can generate genomic, genic and exonic complex rearrangements in humans. Nat Genet 41:849–853.
